Synthesis of steroids



United States Patent 3,316,156 SYNTHESIS OF STEROIDS Saul L. Neidleman,Lawrence Township, Samuel C. Pan, Metuchen, and Patrick A. Diassi,Westtield, N.J., assignors, by mesne assignments, to E. R. Squibb &Sons, Inc., New York, N.Y., a corporation of Delaware N0 Drawing. FiledMar. 26, 1965, Ser. No. 443,080 6 Claims. (Cl. 195-51) ABSTRACT OF THEDISCLOSURE Process for preparing 17oz-hydroxy-16,20-diketo steroids ofthe pregnane series by subjecting the corresponding 16,20-diketosteroids of the pregnane series that are unsubstituted in thel7-position to the action of peroxidase in the presence of hydrogenperoxide under aerobic conditions.

This application is a continuation-in-part of our application, Ser. No.425,077, filed January 12, 1965, and now abandoned.

This invention relates to a new process for preparing17a-hydroxy-l6,20-diketo steroids and to certain new steroids preparedthereby.

The process of this invention essentially comprises subjecting a16,20-diketo steroid of the pregnane series (including the pregnene,pregnadiene and pregnatriene series), unsubstituted in the 17-position,under aerobic conditions to the action of the enzyme peroxidase in thepresence of hydrogen peroxide.

As sources of the peroxidase enzyme, plant cells and saps, animaltissues (such as liver), body fluids (such as saliva), leucocytes(myeloperoxidase), milk (lactoperoxidase) and many microorganisms, suchas Xylaria digitata and Cm'vularz'a lurzata, may be used. The preferredsources of peroxidase for the purpose of this invention are horse-radishand the microorganism, Myroth'ecz'um verrucaria. The peroxidase obtainedfrom the horse-radish can be supplied merely by pressing horse-radishand using the juice obtained or a purified preparation of horse-radishperoxidase may be used. The peroxidase from M. verrucarz'a can beobtained by culturing the microorganism on a suitable nutrient medium,recovering the mycelium formed and treating the mycelium to recoverpurified peroxidase.

In addition to the peroxidase, hydrogen peroxide must also be present inthe reaction mixture. Although hydrogen peroxide itself may be added tothe mixture, preferably the hydrogen peroxide is prepared in situ by useof a peroxide producing enzyme system. Such enzyme systems are wellknown in the art and include glucose oxidase in the presence of glucose,D- and L-amino acid oxidases in the presence of D- or L-methionine, anddiamine oxidase in the presence of histamine. Although substantially anyconcentration of hydrogen peroxide may be used, preferably the hydrogenperoxide is present in a molar ratio of about 0.1 to 1 to about 100 to 1(optimally about 1 to 1 to about to 1) based on the weight of thesteroid. If a peroxide producing enzyme system is used, theconcentration of the enzyme i so adjusted to yield the sameconcentration of hydrogen peroxide as stated above.

The reaction is preferably conducted at a pH in the range of about 4 toabout 7 (optimally about 6 to about 7 and most advantageously at a pH ofabout 6). To assure that the pH of the reaction mixture is maintained inthis range, a buffering agent Which butters in the desired pH range ispreferably also added to the reaction medium. Suitable buffers includeMcIlvaines buifer, potassium citrate buffer, potassium acetate buffer,potassium phosphate buffer and potassium formate butter.

The reaction is carried out in an aqueous medium under aerobicconditions, normally at a temperature in the range of about 20 C. toabout 30 C. The components of the medium, namely, the steroid, bufferingagent, peroxidase and hydrogen peroxide source are merely mixed withWater and the resultant mixture agitated or shaken to assure adequateaeration for about 10 to about 300 minutes (optimally about 30 minutesto about 240 minutes).

Although the peroxidase acts merely as a catalyst and hence can bepresent in any proportion, to assure maximum conversion of the startingsteroid to the desired final product, the peroxidase is present in aweight ratio of about 1 to about 10 (optimally about 2.0) based on thesteroid reactant.

Among the suitable steroid substrates are all steroids of the pregnaneseries that are unsubstituted in the 17- position and contain ketogroups in at least the 16 and 20-positions. Such steroids include16-ketoprogesterone,

I6-keto-A-norprogester0ne,

l6-keto-l-dehydroprogesterone,

16-keto-6-dehydroprogesterone,

16-keto-1,6-tetradehydroprogesterone,

l6-ketopregnenolone,

16-keto-l1-desoxycorticosterone and 21-esters thereof,

6ot-methyl-l6-ketoprogesterone,

6,6-chloro-16-ketoprogesterone,

6a-fluoro-16-ketoprogesterone,

9a-halo-1lfi-hydroxyd6-ketoprogesterones (such as9afluoro-1LB-hydroxy-16-ketoprogesterone),

9a-halo-1 1,16-diketoprogesterones,

9a-halo-1lfi-hydroxy-l6-keto-1-dehydroprogesterones,

9a-halo-1 l B-hydroxy-l 6-keto-6-dehydroprogesterones,

6a,9a-dihalo-l 1 c-hydroxy-l6-ketoprogesterones,

9u-halo-l6-ketocorticosterones and 2l-esters thereof (such as9a-fluoro-lo-ketocorticosterone and its 21-acetate),

9u-halo-l6-keto-l-dehydrocorticosterones and 2l-esters thereof (such as9a-fluoro-l6-keto-1-dehydrocorticosterone and its ZI-acetate),

9oc-halo-16-keto-6-dehydrocortic0sterones and 21-esters thereof,

9a,21-dihalo-1lfl-hydroxy-l6-ketoprogesterones,

911,21 -dihalo-l lfi-hydroxyl 6-keto-1 -dehydropr0gester ones,

9a,21-dihalO-1 l/3-hydroxy-16-keto-6-dehydroprogesterones,

9a-halo-1 1,B-hydroxy-16-keto-A-norprogesterones,

16-keto-19-norprogesterone,

l6-keto-l9-n0rcorticosterone,

6a-halo-16-keto-1 lfl-hydroxycorticosterones and their 21- esters,

and 6a-ha1o-16-keto-1 IB-hydroxy-l-dehydrocorticosterones and their2l-esters.

The products obtained correspond to the steroid substrate but contain a17ot-hydroxy group. Thus, for example, by employing l6-ketoprogesteroneas the substrate, 16-keto-l7a-hydroxyprogesterone, a new steroid of thisinvention having progestational activity and hence useful in lieu ofprogesterone in treatment of conditions for which progesterone is used,is obtained. The other new 17a-hydroxy steroids formed possessglucocorticoid activity if they contain a 11,8-hydroxy or ll-keto group,and progestational activity if there is no substitution in the C-ring.

The following examples illustrate the invention:

EXAMPLE 1 1 6-ket0-1 7 a-hydroxyprogesterione uxidase (Cal. Biochem. No.34641, 1.6 Eu./mg. protein) [1 2 ml. of water, mg. of horse-radishperoxidase :Worthington, Grade D) in 2 ml. of water, 2 ml. of 0.5 V1potassium phosphate bufier (pH 6.0), 5 mg. of 16-ketosrogesterone in 0.4ml. of dimethylsulfoxide and 1.6 ml. )f distilled water and the tubesattached to a rotary ihaker at 25 C. for five hours. The contents oftubes are pooled and extracted twice with one liter of methyl isobutylketone. The combined organic phases are then extracted with water, 5%sodium bicarbonate and water and evaporated to dryness, in vacuo. Theresidue (about 977 mg.) is dissolved in five milliliters of chloroformand the solution applied in equal quantities to twenty-five sheets ofWhatman 3 mm. paper (7.5 wide) and chromatographed using the systemtoluene-propylene glycol with propylene glycol as the stationary phaseand toluene as the mobile phase. The bands at Rf-0.6 which aredetectable under ultraviolet light are cut and eluted with methanol. Themethanol extracts are then evaporated to dryness and the residue takenup in chloroform which is washed with water and evaporated to dryness,in vacuo. Crystallization of the residue from acetone-hexane gives about114.3 mg. of l6-keto-17a-hydroxyprogesterone having a melting pointabout l82-184 C., [071 -90.7 (chloroform),

x- 239 me (6, 18,700),

max

max. @5 53 4.25 (s, 1-11) 7.73 (s, 21Me), 8.98 (S, 19Me), 9.01 (s,18-Me).

Analysis.-Calcd. for C H O (344.44): C, 73.22; H, 8.19. Found: C, 72.99;H, 8.24.

EXAMPLE 2 Following the procedure of Example 1 but substituting the samequantity of L-amino acid oxidase for the glucose oxidase and the samequantity of L-methionine for the glucose, the same product,16-keto-17a-hydroxyprogesterone, is obtained.

cmf

EXAMPLE 3 Following the procedure of Example 1 but substituting the samequantity of diamine oxidase for the glucose oxidase and the samequantity of histamine for the glucose, the same product,16-keto-17a-hydroxyprogesterone, is obtained.

EXAMPLE 4 16-ket0-1 7a-hydroxy-A-norp rogesterone i of the residue fromacetone-hexane or ether gives about 400 mg. ofA-norpregn-3-en-2,l6-20-trione having a melting point of about 128130,[od 44 (chloroform),

M12; 233 mu (6, 18,500), 280 Inn (6, 6200).

Analysis.--Calcd for C H O (314.41): C, 76.40; H, 8.34. Found: C, 76.53;H, 8.31.

(b) Preparation of 16-ke10-17a-hydr0xy-A-n0rpr0gesterone: Following theprocedure of Example 1 but substituting 5 mg. ofA-norpregn-3-en-2,l6,20-trione for the 16 ketoprogesterone,16-keto-l7a-hydroxy-A-norprogesterone having a melting point of about192,

i obtained.

Analysis.-Calcd for C H O (330.41): C, 72.70; H, 7.93. Found: C, 72.77;H, 7.79.

Similarly, any other 17-unsubstituted-l6,20-diketo steroid of thepregnane series may be substituted for the 16- ketoprogesterone in theprocedure of Example 1 to yield the corresponding17zx-hYdfOXY-l6-20-dik6t0 derivative as the product obtained.

This invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A process for preparing a 17u-hydroxy-16,20-diketo steroid of thepregnane series which comprises subjecting a 17-unsubstituted16,20-diketo steroid of the pregnane series under aerobic conditions tothe action of peroxidase in the presence of hydrogen peroxide andrecovering the 17a-hydroxy-l6,20-diketo steroid formed.

2. The process of claim 1, wherein the hydrogen peroxide is prepared insitu by the action of a peroxide producing enzyme system.

3. The process of claim 2, wherein the hydrogen peroxide producingenzyme system is glucose oxidase plus glucose.

4. The process of claim 11 wherein the peroxidase is References Cited bythe Examiner UNITED STATES PATENTS 3,041,250 6/1962 .Wolnok -51 X A.LOUIS MONACELL, Primary Examiner. AL N E.- TA ENHQ TZ. E am n

1. A PROCESS FOR PREPARING A 17A-HYDROXY-16,20-DIKETO STEROID OF THEPREGNANE SERIES WHICH COMPRISES SUBJECTING A 17-UNSUBSTITUTED 16,20-DIKETO STEROID OF THE PREGNANE SERIES UNDER AEROBIC CONDITIONS TO THEACTION OF PEROXIDASE IN THE PRESENCE OF HYDROGEN PEROXIDE AND RECOVERINGTHE 17 A-HYDROXY-16, 20-DIKETO STEROID FORMED.